1. Field of the Invention
The present invention relates to microbial enzymes capable of degrading the rhamnogalacturonan backbone in hairy regions of pectins, more specifically to novel families of microbial rhamnogalacturonan hydrolases and the genes encoding such enzymes; to a method of producing such enzymes; and to methods for using such enzymes in the textile, detergent, animal feed and cellulose fiber processing industries.
2. Description of the Related Art
Pectic polysaccharides constitute the major matrix polysaccharides in the middle lamella and primary cell wall of dicotyledonous plants (Carpita and Gibeaut, 1993). The main backbone in pectins can be divided into linear homogalacturonan (smooth) regions of up to 200 residues of (1,4)-linked alpha-D-galacturonic acid (GalUA), and highly branched rhamnogalacturonan (hairy) regions consisting of a backbone of repeating alpha-(1,2)-L-Rha-alpha-(1,4)-D-GalUA disaccharide units (Carpita and Gibeaut, 1993; O""Neill et al., 1990; Thibault et al., 1993). The hydroxyl at the C-4 position of the rhamnose residues serves as the attachment point for the side chains (hairs), consisting mainly of neutral oligosaccharides, such as arabinan, galactan and/or arabinogalactan (Carpita and Gibeaut, 1993; O""Neill et al., 1990; Schols et al., 1990). In addition, the GalUA residues in the backbone may be acetyl esterified at the C-2 or C-3 position or methyl esterified at the carboxy group (Carpita and Gibeaut, 1993; Schols et al., 1990).
The distribution and composition of the side chains vary considerably between different cell types and physiological states, but in general about half of the rhamnosyl units in the rhamnogalacturonan regions have side chains attached. The galactan side chains are in most plants type 1 galactans, which are composed of xcex2-1,4 linked galactopyranose with some branching points and a length of up to 60 saccharide units (DP60). Arabinofuranose residues or short arabinan oligomers can be attached to the galactan chain at the o-3 of the galactosyl unit, thus named arabinogalactan. Galactans (or arabinogalactans) have an important function in the primary cell wall, where they interact with other structural components of the cell wall such as xyloglucans or arabinoxylans. Thus they possibly serve to anchor the pectic matrix in the cell wall. (Carpita and Gibeaut, 1993, Plant J., 3, 1-30; O""Neill et al., 1990, Methods in Plant Biochemistry, 415-441; Selvendran, 1983, The Chemistry of Plant Cell Walls. Dietary Fibers; Hwang et al., Food Hydrocolloids, 7, 39-53; Fry, 1988, The growing Plant Cell Wall: Chemical and Metabolic Analysis).
Sugar beet debranched arabinan and potato galactan from Megazyme (Ireland, http://www.megazyme.com/Purchase/index.html) contain rhamnose and galacturonic acid indicating that these substrates and their AZCL derivatives contain some rhamnogalacturonan.
The biological degradation of pectic substances is a complex process involving several enzymes produced by a wide variety of saprophytic, plant pathogenic fungi and bacteria (Pilnik and Rombouts, 1979). For example, the hydrolysis of smooth, homogalacturonan regions of pectin by polygalacturonases is dependent upon demethylation of the homogalacturonan backbone by pectin methylesterase (Christgau et al., 1996; Pilnik and Rombouts, 1979). Several microbial polygalacturonases, pectate lyases, pectin methylesterases and pectin lyases active within the smooth regions of pectin have been described in literature.
A number of enzymes capable of hydrolyzing arabinan, galactan or arabinogalactan side chains in the hairy regions have been characterized. By contrast, only few enzymes capable of degrading the rhamnogalacturonan backbone have been reported. A rhamnogalacturonan hydrolase belonging to family 28 of glycosyl hydrolases and a rhamnogalacturonan lyase belonging to lyase family 4, both from Aspergillus aculeatus, have been cloned and characterized (Kofod et al. (1994) Journal of Biological Chemistry Vol. 269 (46) pp. 29182-29189; Kauppinen et al. (1995) Journal of Biological Chemistry Vol. 270 (45) pp. 27172-27178; Azadi et al. (1995) Glycobiology Vol. 5 (8) pp. 783-789; Mutter et al. (1996) Plant Physiology Vol. 110 (1) pp. 73-77; Mutter et al. (1998) Plant Physiology Vol. 117 (1) pp. 141-152; Mutter et al. (1998) Carbohydrate Research Vol. 311 (3) pp. 155-164). The sequence families can be found on http://afmb.cnrs-mrs.fr/xcx9cpedro/CAZY/db.html.
Degradation of rhamnogalacturonan by the Aspergillus rhamnogalacturonan hydrolase and lyase is enhanced by removal of acetyl groups from the backbone (Kofod et al., 1994; Schols et al., 1990). A rhamnogalacturonan acetylesterase (RGAE) cloned and characterized from Aspergillus aculeatus specifically removes acetyl groups from hairy regions and acts in synergy with the rhamnogalacturonases in degradation of apple pectin rhamnogalacturonan (Kauppinen et al., 1995).
JP 10-033169 discloses a method for purification of rhamnogalacturonase from enzyme preparations containing numerous enzymes or liquid cultures of Aspergillus, Bacillus or Erwinia.
The object of the present invention is to provide a novel rhamnogalacturonan hydrolase enzyme which can degrade the backbone of hairy regions of pectin in an effective manner useful in a number of different industrial applications.
The inventors have now found a number of novel bacterial enzymes exhibiting rhamnogalacturonan hydrolase activity which are believed to be members of two hitherto unidentified families of glycosyl hydrolases according to the classification based on hydrophobic cluster analysis (Henrissat, B. et al.). The novel enzymes have no amino acid sequence homology to known rhamnogalacturonan hydrolases from family 28 or rhamnogalacturonan lyases from lyase family 4. More specifically, novel families of enzymes degrading rhamnogalacturonan by hydrolysis has been found. The enzymes are of bacterial origin, with few or no cystein bridges and with a potential of being expressed in high yields in Gram positive bacterial hosts. The rhamnogalacturonase enzymes of this invention show enzymatic activity at neutral and alkaline conditions and, accordingly, they are very useful in a number of industrial applications.
The novel enzymes exhibit catalytic activity on rhamnogalacturonan as well as on the Megazyme products AZCL potato galactan and AZCL debranched arabinan. This activity can be explained by the presence of rhamnogalacturonan in the two AZCL carbohydrate polymers.
The inventors have succeeded in identifying either partial or full length DNA sequences encoding the novel enzymes. The DNA sequences are listed in the appended sequence listing as SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19, and the deduced amino acid sequences are listed in the sequence listing as SEQ ID NOS: 2, 4, 6, 8, 10, 12 ,14, 16, 18 and 20, respectively.
In a first aspect, the present invention relates to an enzyme exhibiting rhamnogalacturonan hydrolase activity wherein the enzyme belongs to a glycosyl hydrolase family other than family 28. In a preferred embodiment, the enzyme of the invention is obtained from a microbial strain belonging to Bacteria, preferably to Firmicutes or Proteobacteria, more preferably to Actinobacteria, Myxobacteria or the Bacillus/Clostridium group.
In second and third aspects, the invention relates to an enzyme comprising at least one amino acid sequence segment selected from the group of amino acid sequence segments consisting of NIRAGAHTQF(M or L)VYD(F or L)DGDGKAE (SEQ ID NO:21); YGNRVDRFLAG (SEQ ID NOS: 22); YGNRVDRFLAGXAYLDG (SEQ ID NO:23); AGQGNH(N or S)LS(I or V)ADVDGDGKDEII (SEQ ID NO:24); and AGQGNH(N or S)L(S or A)(I or V)ADVDGDGKDEII (SEQ ID NO:25); and to an enzyme comprising at least one amino acid sequence segment selected from the group of amino acid sequence segments consisting of EVRDATIGLL (SEQ ID NO:26); NNYVVGNPI (SEQ ID NO:27); and DADRTNRA (SEQ ID NO:28).
In further aspects, the invention relates to a rhamnogalacturonan hydrolase enzyme which is i) a polypeptide produced by a strain selected from the group consisting of Bacillus licheniformis, Bacillus halodurans, Bacillus subtilis, Bacillus agaradhaerens, Bacillus sp. AA386, Sorangium cellulosum, Streptomyces coelicolor and Caldicellulosiruptor sp.; or ii) a polypeptide comprising an amino acid sequence as shown in positions 1-621 of SEQ ID NO:2 or in positions 1-620 of SEQ ID NO:4, or in positions 1-620 of SEQ ID NO:6 or in positions 1-471 of SEQ ID NO:8 or in positions 1-170 of SEQ ID NO:10 or in positions 1-112 of SEQ ID NO:12 or in positions 1-655 of SEQ ID NO:14 or in positions 1-631 of SEQ ID NO:16 or in positions 1-389 of SEQ ID NO:18 or in positions 1-169 of SEQ ID NO:20; or iii) an analogue of the polypeptide defined in i) or ii) which is at least 75% homologous with said polypeptide and can be derived from said polypeptide by substitution, deletion or insertion of one or several amino acids.
Within other aspects, the present invention provides an isolated polynucleotide molecule selected from the group consisting of (a) polynucleotide molecules encoding a rhamnogalacturonase and comprising a sequence of nucleotides iselected from the group consisting of the nucleotide sequences shown in SEQ ID NO: 1 from nucleotide 1 to nucleotide 1863, SEQ ID NO:3 from nucleotide 1 to nucleotide 1863, and SEQ ID NO:5 from nucleotide 1 to nucleotide 1863, in SEQ ID NO: 7 from nucleotide 1 to nucleotide 1413, in SEQ ID NO: 9 from nucleotide 1 to nucleotide 512, in SEQ ID NO: 11 from nucleotide 1 to nucleotide 336, in SEQ ID NO: 13 from nucleotide 1 to nucleotide 1965, in SEQ ID NO: 15 from nucleotide 1 to nucleotide 1896, in SEQ ID NO: 17 from nucleotide 1 to nucleotide 1168, in SEQ ID NO: 19 from nucleotide 1 to nucleotide 507; (b) species homologs of (a); (c) polynucleotide molecules that encode a polypeptide which can degrade the rhamnogalacturonan backbone of hairy regions of pectin and which is at least 75% identical to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 1 to amino acid residue 621, SEQ ID NO: 4 from amino acid residue 1 to amino acid residue 620, or SEQ ID NO: 6 from amino acid residue 1 to amino acid residue 620, SEQ ID NO: 8 from amino acid residue 1 to amino acid residue 471, SEQ ID NO: 10 from amino acid residue 1 to amino acid residue 170, SEQ ID NO: 12 from amino acid residue 1 to amino acid residue 112, SEQ ID NO: 14 from amino acid residue 1 to amino acid residue 655, SEQ ID NO: 16 from amino acid residue 1 to amino acid residue 631, SEQ ID NO: 18 from amino acid residue 1 to amino acid residue 389, SEQ ID NO: 20 from amino acid residue 1 to amino acid residue 169; (d) molecules complementary to (a), (b) or (c); and (e) degenerate nucleotide sequences of (a), (b), (c) or (d).
The E. coli plasmids comprising the polynucleotide molecules (the DNA sequences corresponding to SEQ ID NOS: 1, 3, 9, 13, 17 respectively) encoding an enzyme of the present invention has been transformed into a strain of the Escherichia coli which was deposited by the inventors according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Federal Republic of Germany, on Apr. 24, 1998 under the deposition numbers DSM 12123 and DSM 12122, on Sep. 8, 1998 under the deposition number DSM 12405, on Apr. 24, 1998 under the deposition number DSM 12124, and on May 29, 1998 under the deposition number DSM 12202, respectively.
Within another aspect of the invention there is provided an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment selected from the group consisting of (a) polynucleotide molecules encoding a rhamnogalacturonan hydrolase and comprising a sequence of nucleotides as shown in SEQ ID NO: 1 from nucleotide 1 to nucleotide 1863, SEQ ID NO:3 from nucleotide 1 to nucleotide 1863, and SEQ ID NO:5 from nucleotide 1 to nucleotide 1863, in SEQ ID NO: 7 from nucleotide 1 to nucleotide 1413, in SEQ ID NO: 9 from nucleotide 1 to nucleotide 512, in SEQ ID NO: 11 from nucleotide 1 to nucleotide 336, in SEQ ID NO: 13 from nucleotide 1 to nucleotide 1965, in SEQ ID NO: 15 from nucleotide 1 to nucleotide 1896, in SEQ ID NO: 17 from nucleotide 1 to nucleotide 1168, in SEQ ID NO: 19 from nucleotide 1 to nucleotide 507; (b) species homologs of (a); (c) polynucleotide molecules that encode a polypeptide which can degrade rhamnogalacturonan backbone of hairy regions of pectin and which is at least 75% identical to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 1 to amino acid residue 621, SEQ ID NO: 4 from amino acid residue 1 to amino acid residue 620, or SEQ ID NO: 6 from amino acid residue 1 to amino acid residue 620, SEQ ID NO: 8 from amino acid residue 1 to amino acid residue 471, SEQ ID NO: 10 from amino acid residue 1 to amino acid residue 170, SEQ ID NO: 12 from amino acid residue 1 to amino acid residue 112, SEQ ID NO: 14 from amino acid residue 1 to amino acid residue 655, SEQ ID NO: 16 from amino acid residue 1 to amino acid residue 631, SEQ ID NO: 18 from amino acid residue 1 to amino acid residue 389, SEQ ID NO: 20 from amino acid residue 1 to amino acid residue 169; and (d) degenerate nucleotide sequences of (a), (b), or (c); and a transcription terminator.
Within yet another aspect of the present invention there is provided a cultured cell into which has been introduced an expression vector as disclosed above, wherein said cell expresses the polypeptide encoded by the DNA segment.
Within another aspect of the present invention there is provided a composition comprising a purified polypeptide according to the invention, i.e. an enzyme, in combination with other polypeptides exhibiting enzymatic activity.
At present it is contemplated that the novel enzyme of the present invention is useful for the treatment of cellulosic material, especially cellulose-containing fiber, yarn, woven or non-woven fabric. The treatment can be carried out during the processing of cellulosic material into a material ready for garment manufacture or fabric manufacture, e.g. in the desizing or scouring step; or during industrial or household laundering of such fabric or garment.
Accordingly, in further aspects the present invention relates to a detergent composition comprising a rhamnoglacturonan hydrolase enzyme or an enzyme capable of degrading rhamnogalacturonan backbone of hairy regions of pectin; and to use of the enzyme of the invention for the treatment of cellulose-containing fibers, yarn, woven or non-woven fabric.
It is also contemplated that the enzyme of the invention is effective for use in an enzymatic scouring process in the preparation of cellulosic material e.g. for proper response in subsequent dyeing operations. Further, it is contemplated that detergent compositions comprising the novel enzyme are capable of removing or bleaching certain soils or stains present on laundry, e.g. soils and spots resulting from food, plants, and the like containing pectic substances. It is also contemplated that treatment with detergent compositions comprising the novel enzyme can prevent binding of certain soils to the cellulosic material.